How epigenetic states are inherited during S phase of the cell cycle is one of the most challenging questions in the chromatin and epigenetic fields. DNA replication-coupled nucleosome assembly plays an important role in epigenetic inheritance following DNA replication and DNA repair. Mutations of most, if not all, genes involved in replication-coupled nucleosome assembly result in defects in transcriptional silencing at heterochromatin and genome instability in both yeast and mammalian cells. We have been studying how nucleosomes are formed following DNA replication in yeast and human cells and have made multiple significant contributions to this process. However, how parental histone (H3-H4)2 tetramers are transferred to replicating DNA is still poorly understood, which hinders our understanding of transmission of epigenetic information into daughter cells. The major challenge to understanding parental histone (H3-H4)2 assembly is a lack of methods to track this process. Despite this challenge, we have developed the eSPAN (enrichment and Sequencing Protein- Associated Nascent DNA) method that can discern whether a protein binds to leading or lagging strands of DNA replication forks. This method enables us for the first time to monitor nucleosome assembly of both newly synthesized and parental histone (H3-H4)2 onto leading and lagging strands of DNA replication forks. In this proposal, we will elucidate molecular mechanisms whereby parental (H3-H4)2 are assembled into nucleosomes following DNA replication and how epigenetic marks are inherited during mitotic cell division in both yeast and human cells using a combination of genetic, biochemical and genomic approaches. Together, our studies should have a profound impact on the understanding of nucleosome assembly and epigenetic inheritance.